This proposal is focused on vinculin, a cytoskeletal protein that is a prominent component of focal adhesions and adherens junctions. Vinculin is critical for cell migration, cell survival and embryogenesis and disruption of vinculin expression in mice results in an embryonic lethal phenotype. Vinculin functions as a mechanical linker between transmembrane integrin receptors and the actin cytoskeleton through association with cell adhesion molecules. Release of autoinhibitory head/tail interactions is believed to activate vinculin's function as a scaffold protein to facilitate assembly of multi-protein networks important for modulation of cytoskeleton structure and for downstream signaling. Although vinculin binds to a large number of cytoskeletal proteins, in many cases, we do not understand the nature of the interaction or functional significance. For example, the role of paxillin interactions and with the vinculin tail (Vt) domain in vinculin function is not known. In addition, several functional roles for acidic phospholipids (PL) have been proposed, including the involvement of acidic PLs in the activation process as well in regulating vinculin function once vinculin is activated. While recent studies suggest that vinculin/PL interactions promote uncoupling of adhesion sites from the actin cytoskeleton, our preliminary data indicates that these studies were conducted with mutants that are likely to multiple defects, including PL binding. Thus, while acidic PLs, specifically inositol 4, 5 bisphosphate, is believed to play an important role in vinculin regulation and function, its role remains unclear. Our efforts will focus on NMR, biophysical, biochemical and cell biological studies of vinculin, with the primarily focus on the role of ligand interactions with Vt. We will investigate the structural features of Vt, interactions of wt Vt with acidic PLs and paxillin, and explore the importance of these interactions in the biological function of vinculin. The specific aims of this proposal are to investigate the: 1) Regulation of vinculin through PL binding. 2) Structural basis for Vt/paxillin complex formation and the role of these interactions in controlling vinculin function. PUBLIC HEALTH RELEVANCE Vinculin is an essential protein involved controlling cell shape and motility, in part by anchoring actin polymers to the membrane. This function also extends to cancer cells, by regulating their movement and proliferation to other parts of the body. Hence, not too surprising, vinculin exhibits properties of a tumor suppressor protein. The activity of vinculin is controlled by regulating auto-inhibitory or `self'interactions between its head and tail domain. The goal of this effort is to characterize tail domain interactions with select proteins and lipids, to better understand how these binding interactions regulate vinculin activity and function. Results obtained from these studies may aid in developing novel therapeutic approaches for counteracting aberrant vinculin activity in human disease.